Digestion, Fermentation, and Pathogen Anti-Adhesive Properties of the Hmo-Mimic Di

bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399972; this version posted November 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Digestion, fermentation, and pathogen anti-adhesive properties of the hMO-mimic di-

fucosyl-β-cyclodextrin

Stella A. Verkhnyatskaya,a,† Chunli Kong,b,† Cynthia Klostermann,c,† Henk A. Schols,d Paul de Vos,b

Marthe T.C. Walvoorta,*

a Stratingh Institute for Chemistry, University of Groningen, Groningen, the Netherlands

b Department of Pathology and Medical Biology, University Medical Center Groningen, Groningen, the

Netherlands

c Biobased Chemistry and Technology, Wageningen University & Research, Wageningen, the

Netherlands

d Laboratory of Food Chemistry, Wageningen University & Research, Wageningen, the Netherlands

† Authors contributed equally

Keywords: human milk oligosaccharides, L-fucose, cyclodextrin, anti-adhesive, digestion

Correspondence: Dr. Marthe T.C. Walvoort, [email protected]

List of abbreviations: βCD, β-cyclodextrin; DFβCD, di-Fuc-β-cyclodextrin; DFL, 2’,3-difucosyllactose;

EHEC, enterohemorrhagic E. coli; EPEC, enteropathogenic E. coli; ETEC, enterotoxigenic E. coli; 2’-

FL, 2’-fucosyllactose; 3-FL, 3-fucosyllactose; FOS, fructo-oligosaccharides; Fuc, L-fucose; Gal, D-

galactose; Glc, D-glucose; GlcNAc, N-acetyl-D-glucosamine; GOS, galacto-oligosaccharides; hMO,

human milk oligosaccharide; MFβCD, mono-Fuc-β-cyclodextrin; NDC, non-digestible carbohydrate;

Sia, D-sialic acid; 6’-SL, 6’-sialyllactose

1 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399972; this version posted November 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Abstract

Scope: Human milk is widely acknowledged as the best food for infants, and that is not just because of

nutritional features. Human milk also contains a plethora of bioactive molecules, including a large set

of human milk oligosaccharides (hMOs). Especially fucosylated hMOs have received attention for their

anti-adhesive effects on pathogens by preventing attachment to the intestinal wall. Because hMOs are

generally challenging to produce in sufficient quantities to study and ultimately apply in (medical) infant

formula, hMO mimics are interesting compounds to produce and evaluate for their biological effects.

Methods and results: We investigated the digestion, fermentation, and pathogen anti-adhesive capacity

of the novel hMO mimic di-fucosyl-β-cyclodextrin (DFβCD). We establish that DFβCD is not digested

by α-amylase and also resists fermentation by the microbiota from a 9 month-old infant. In addition, we

reveal that DFβCD blocks adhesion of enterotoxigenic E. coli (ETEC) to Caco-2 cells, especially when

DFβCD is pre-incubated with ETEC prior to addition to the Caco-2 cells.

Conclusion: Our results suggests that DFβCD functions through a decoy effect. We expect that our

results inspire the generation and biological evaluation of other fucosylated hMOs and mimics, to obtain

a comprehensive overview of the anti-adhesive power of fucosylated glycans.

2 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399972; this version posted November 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1. Introduction

Human milk is generally considered to be the best nutritional source for infants, as an increasing amount

of research reveals the health effects of a plethora of bioactive components in human milk, including

beneficial microbes,[1] immunoglobulins,[2] and human milk oligosaccharides (hMOs).[3, 4]

Especially the hMOs have received considerable attention as health-promoting factors over the last

decades, as they are shown to affect the development of the infant’s microbiome, promote immune

system maturation, and ward off infections by acting as decoy substrates, amongst other effects.[5]

Interestingly, hMOs are virtually absent from bovine milk-based infant formulas [6], and as a result,

there is a broad interest to produce hMOs with the aim to add them to infant and medical formula. To

achieve this goal, researchers are investigating the mode of action of specific hMOs to unravel structure-

activity relationships that will guide the future selection of health-promoting hMO-based additives.

Simultaneously, procedures are being developed to generate specific hMOs using (chemo)enzymatic

and microbial cell factory approaches.[7] This has resulted in the recent approval by both the U.S. Food

and Drug Administration (FDA) and the European Union of two short hMO structures, i.e. 2’-

fucosyllactose (2’-FL) and lacto-N-neotetraose (LNnT), that are currently added to certain brands of

infant formula.[8, 9]

Currently, more than 200 different hMO structures have been identified in human milk [10] and

although they together form a complex mixture of carbohydrates, they do share certain structural

similarities. HMOs are generally composed of a linear or branched backbone containing alternating N-

acetyl--D-glucosamine (GlcNAc) and -D-galactose (Gal) building blocks, capped with a lactose

disaccharide on the reducing end. At specific positions these backbones can be decorated with -L-

fucose (Fuc) and -D-neuraminic acid, also called sialic acid (Sia).[5] Comparing both types of

decorations, it is interesting to note that Fuc is much more prevalent (on 50-80% of the hMOs) than Sia

(on maximum 30% of hMOs).[11] Fucosylated hMOs have been linked to numerous health effects,

including correcting microbial dysbiosis in cesarean-born infants[12] and protection against viral and

bacterial infections[13] and bacterial gastroenteric infections.[14] Especially the anti-adhesive activity

of fucosylated hMOs on pathogens is striking and reported by many.[15, 16] One of the first examples

3 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399972; this version posted November 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

of the anti-adhesive potential of hMOs was published in 1990, where the neutral fraction (i.e. not

containing Sia building blocks) of isolated hMOs showed a significant reduction in the adhesion of

enteropathogenic E. coli (EPEC) and a concomitant preventative effect on the development of urinary

tract infections.[17] In addition, maternal secretor status, which directly impacts the final structure and

abundance of fucosylated oligosaccharide levels in the milk, was linked to infection rates and symptom

severity in their offspring.[18] Using bioengineered samples of 2’-FL and 3-fucosyllactose (3-FL, both

shown in Figure 1), moderate anti-adhesive effects against Pseudomonas aeruginosa, Campylobacteri

jejuni, EPEC, and Salmonella enterica serovar fyris on intestinal Caco-2 cells were established.[19, 20]

Moreover, 2’-FL was also found to block adhesion of E. coli O157 (an enterohemorrhagic E. coli strain,

EHEC) onto intestinal Caco-2 cells.[21] Because the structural complexity of hMOs prevents their

straightforward production in sufficient quantities to add to infant formula, structural analogs with

similar functions have been developed, and with great success. Galacto-oligosaccharides (GOS) and

fructo-oligosaccharides (FOS) are regularly added to infant formula, and induce especially the

development of a healthy microbiome.[22, 23] Other so-called non-digestible carbohydrates (NDCs)

include pectins, chitin and chitosan, alginates, and mannans, and many of these polysaccharides have

been revealed to have anti-pathogenic effects. [24] As these NDCs are structurally highly different from

native hMOs, this showcases that novel carbohydrate structures have the potential to mimic the

beneficial effects of hMOs, without the challenging production that hMOs would require.

All this withstanding, the production of novel hMO analogs with enzymatic methods is currently

an active area of research.[25] Enzymatic methods are currently mostly focused on hMOs containing

either no or one decorative Fuc or Sia, including 2’-FL and 6’-sialyllactose (6’-SL).[26] With respect to

fucosylated hMOs, details are emerging pertaining to the degree of backbone fucosylation and

positioning on the backbone needed for a specific effect. For instance, 2’,3-di-fucosyllactose (DFL)

exhibited a stronger antimicrobial effect against Streptococcus agalactiae GB590 than mono-

fucosylated lactoses 2’-FL and 3-FL.[27] Especially a higher degree of fucosylation is nearly impossible

to obtain with current enzymatic methods, compromising the ability to unravel the impact of higher

degrees of fucosylation on anti-adhesion activity. Therefore, we recently developed a chemical strategy

4 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399972; this version posted November 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

to produce di-fucosylated β-cyclodextrin (DFβCD, Figure 1).[28] The family of cyclodextrins has the

GRAS status (Generally Recognized As Safe), and they are frequently used in medical formulations and

as food additives.[29] -Cyclodextrin (CD) is shown in Figure 1 and contains seven glucose (Glc)

residues linked in an -1,4 fashion, analogous to the structural composition of maltooligosaccharides.

Using appropriately functionalized β-cyclodextrin and fucose building blocks, the di-fucosylated

DFβCD was obtained in a highly regiospecific manner using chemical strategies.[28] Since the Fuc

moieties are connected to the O-3 position of the backbone glucoses, the decorative pattern mimics the

pattern in 3-FL (Figure 1), which also contains a Fuc moiety that is -1,3 linked to glucose. As a result

of this straightforward approach, DFβCD was produced in sufficient quantities (~ 0.5 g) to test its

functional activity, and to determine whether this structural hMO mimic also is a functional hMO mimic.

Here we present our in-depth studies into the digestion and fermentation of DFβCD, as well as

its anti-adhesive properties against enterotoxigenic Escherichia coli (ETEC) O78:H11. ETEC is the

most common bacterial cause of diarrhea in children in developing countries, and although adhesion of

ETEC to host cells is an intricate combination of factors,[30] it has been established that ETEC adhesion

is also mediated by binding of bacterial lectins to host glycans.[31] In addition, 2’-FL was shown to

reduce adhesion and invasion of ETEC bacteria to T84 intestinal epithelial cells in vitro.[32] Our results

reveal that DFβCD is not digested and fermented by a 9 month-old infant’s inoculum, whereas it does

have anti-adhesive properties against ETEC. This suggests that also hMO analogs such as DFβCD,

composed of a different backbone structure but displaying appropriately spaced decorative Fuc moieties,

can have similar health-beneficial effects as hMOs.

Figure 1. Overview of the molecules used in this study.

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2. Experimental Section

2.1 Materials

The human milk oligosaccharides 2’-fucosyllactose (2’-FL) and 3-fucosyllactose (3-FL) were provided

by Elicityl (France). Difucosylated β-cyclodextrin (DFβCD) was chemically synthesized based on the

commercially obtained β-cyclodextrin (βCD), as described previously, and monofucosylated β-

cyclodextrin (MFβCD) was isolated as a side product.[28] Pancreatin from porcine pancreas (containing

amylase, lipase, and protease) and amyloglucosidase (260 U/mL) were obtained from Sigma-Aldrich

(St. Louis, MO, USA). All materials needed for preparation of SIEM medium were obtained from

Tritium Microbiology. E. coli ET8 was a gift from Prof. Gilles van Wezel (Leiden University)[33] and

E. coli O78:H11 (ATCC35401) was purchased from ATCC. All chemicals used were of analytical

grade.

2.2 In vitro digestibility of di-Fuc-β-cyclodextrin and β-cyclodextrin

Digestion was performed according to Martens et al. with minor modifications.[34] Di-Fuc-β-

cyclodextrin (DFβCD) and β-cyclodextrin (βCD) were suspended in 100 mM sodium acetate buffer pH

5.9. Pancreatin solution was prepared according to Martens et al. [34], without the addition of invertase.

In short, 150 mg pancreatin was suspended in 1 mL MQ and mixed for 10 min. The suspension was

centrifuged for 10 min at 4 °C, 1500 x g. The final enzyme mixture was prepared by mixing 610 µL

pancreatin supernatant with 58 µL amyloglucosidase and 83 µL MQ. Samples were incubated with 200

µL enzyme mixture for 0, 20, 60, 120, and 240 min at 20 mg/mL substrate concentration and enzymes

were inactivated by boiling the sample for 15 min at 100 °C. Released glucose content was analysed

with the GOPOD assay from Megazyme (Grey, Ireland).

2.3 In vitro fermentation of di-Fuc-β-cyclodextrin, β-cyclodextrin, 2’-FL and 3-FL

Fecal sample from one 9 month-old infant (vaginally born, breast-fed, introduced to solid food, no

administration of antibiotics, exposed to probiotic Bifidobacteria) was collected and immediately stored

in an anaerobic container. Fecal slurry was prepared by mixing fresh feces with a pre-reduced dialysate-

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glycerol solution according to Aguirre et al.[35] at 25 % w/v feces dialysate-10 % glycerol. The fecal

slurry was snap-frozen in liquid nitrogen and stored at -80 °C. Standard Illeal Efflux Medium (SIEM)

was prepared according to Logtenberg et al.[36] with minor modifications. The carbohydrate medium

component contained (g/L): pectin, 12; xylan, 12; arabinogalactan, 12; amylopectin, 12; and starch, 100,

with a final concentration of 0.592 g/L. The salt medium component contained 0.144 g/L NaCl. The

inoculum was prepared by diluting the fecal slurry 25 times in SIEM medium. The substrate of interest

was dissolved in SIEM medium at 2.22 mg/mL and 10 % inoculum was added. The in vitro fermentation

was performed in the anaerobic chamber using sterile serum bottles. Samples were inoculated in

duplicate and incubated for 0, 4, 8, 12, 24, and 36 h. In addition, blanks without substrate or without

inoculum were incubated too. At each time point, 200 µL sample was taken from each bottle with a

syringe. This sample was heated for 10 min at 100 °C to inactivate the enzymes and stored at -20 °C

until analysis.

2.4 Substrate degradation analysis

Samples were diluted to 20 µg/mL (DFβCD and βCD) or 10 µg/mL (2’-FL and 3-FL) and centrifuged

at 19000 x g for 10 min. The supernatant (10 µL injection volume) was analyzed using an ICS 3500

HPAEC system from Dionex (Sunnyvale, USA), in combination with a CarboPac PA-1 (2 x 250 mm)

column, with a Carbopac PA-1 guard column (Dionex). Carbohydrate peaks were detected by an

electrochemical Pulsed Amperometric detector (Dionex) after elution with 0.3 mL/min at 25 °C. The

eluents consisted of A (0.1 M NaOH solution) and B (1 M NaOAc in 0.1 M NaOH). Two different

gradients were used. For DFβCD and βCD the gradient used was: 2.5-25 % B (0-30 min), 25-100 % B

(30-40 min), 100 % B (40-45 min), 2.5 % B (45-60 min). For 2’-FL and 3-FL the gradient used was 0-

15 % B (0-15 min), 15-100 % B (15-20 min), 100 % B (20-25 min), 0 % B (25-45 min). Substrates were

quantified using 2.5-10 µg/mL 2’-FL and 3-FL or 5-20 µg/mL DFβCD and βCD. In addition, mono-

Fuc-β-CD (MFβCD) was injected as a pure compound to detect it within the DFβCD mix. Data analysis

was performed with ChromeleonTM 7.2.6 software from Thermo Fisher Scientific (Waltham,

Massachusetts, USA).

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2.5 Organic acid formation after in vitro fermentation

Samples were diluted 5 times and centrifuged at 19000 x g for 10 min. The supernatant (10 µL injection

volume) was analyzed using an Ultimate 3000 HPLC system from Dionex, in combination with an

Animex HPX-87H column (Bio-Rad laboratories Inc, Hercules, USA). Samples were detected by a

refractive index detector (RI-101, Shodex, Yokohama, Japan) and a UV detector set at 210 nm (Dionex

Ultimate 3000 RS variable wavelength detector). Elution was performed at 0.5 mL/min and 50 °C using

50 mM sulphuric acid as eluent. Acetate, propionate, butyrate, lactate, and succinate standard curves

were used for quantification (0.05 – 2 mg/mL). Data analysis was performed with ChromeleonTM 7.2.6

software from Thermo Fisher Scientific.

2.6 Culturing of the intestinal epithelial cell line

Human intestinal epithelial Caco-2 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM,

Lonza), supplemented with 0.5% penicillin-streptomycin (50 μg/mL-50 μg/mL, Sigma), 1% non-

essential amino acid (100x, Sigma), 10mM HEPES (Sigma), and 10% heat deactivated fetal calf serum

(Invitrogen). Caco-2 cells between passage number of 15-20 were chosen for the experiment, and cells

4 were routinely cultured in a humidified incubator with 5% CO2, at 37 ℃. A number of 3×10 Caco-2

cells were seeded onto 24-well plates and cultured for 21 days before use.

2.7 Culturing of bacterial cells

Pathogenic bacteria of Escherichia (E.) coli ET8, and E. coli O78: H11 were recovered from glycerol

stocks at -80 ℃ overnight, and were cultured in Brain heart infusion (BHI). After recovery culture, E.

coli ET8 and E. coli O78: H11 were plated on BHI agar. A single colony of each bacterium was

inoculated from the agar plates to BHI broth for a second overnight culture at 37 ℃ before the adhesion

assay.

2.8 Bacterial adhesion assay

All compounds were dissolved into 2, 5, and 10 mg/mL in antibiotics-free cell culture medium with 1%

dimethyl sulfoxide. The cell culture medium without the tested molecules served as control. All

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compounds were heated for 30 min at 65 ℃ to remove any endotoxin contamination before use. We

first tested the concentration-dependent effect of the molecules on the adhesion of one pathogen E. coli

ET8 to Caco-2 cells. This was done by pre-incubation of Caco-2 cells with 2’-FL, 3-FL, βCD, and

DFβCD of 2, 5, and 10 mg/mL for 2h, respectively. E. coli ET8 was collected after 2h of culture with

centrifugation at 2000 x g for 10 min and washed one time with PBS. The optical density (OD) of E.

coli ET8 was adjusted to OD540 = 0.6 in PBS, and re-suspended either with or without the molecules at

different concentrations. After that Caco-2 cells were co-incubated with the pathogens for another 2h at

37 ℃. Afterwards, the non-adherent bacteria were washed away for three times in PBS. The adherent

bacteria were released in 200μL of 0.1% Triton-X100, and underwent serial dilutions in PBS. The drop-

plating method [37] was employed to plate the adherent bacteria on BHI agar plates (n = 3). Then the

molecules were applied at a concentration of 10 mg/mL to test the influence of the molecules on the

adhesion of E. coli O78: H11 to Caco-2 cells. Here, to test the possible effects of the molecules on the

adhesion, we either pre-incubated the molecules with Caco-2 cells to explore whether they could

influence the bacteria adhesion through modification of the receptors on gut epithelial cells, or we pre-

incubated the molecules with the bacteria to determine a possible decoy effect on the bacteria.

Pre-incubate with Caco-2 cells. As performed with E. coli ET8, all molecules at 10 mg/mL were pre-

incubated with Caco-2 cells for 24h. E. coli O78: H11 was collected after 2h of subculture, when a log

phase was reached, by centrifugation at 2000 x g for 10 min and the cells were washed with PBS. The

OD of E. coli O78: H11 was adjusted to OD540 = 0.6 in PBS, and re-suspended either with or without

the molecules and infected Caco-2 cells for another 2h at 37 ℃.

Pre-incubate with bacteria. E. coli O78: H11 was collected as described above, pre-incubated with the

molecules for 2h at 37 ℃ after re-suspension, and then infected Caco-2 cells for another 2h at 37 ℃.

After infection, Caco-2 cells were gently washed three times to remove the non-adherent bacteria, and

the adherent bacteria were collected with 200 μL of 0.1% Triton-X100, followed by serial dilutions in

PBS. Total colony-forming units (CFUs) were determined after drop-plating method (n = 5).

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2.9 Statistical analysis

Statistical analysis was performed with GraphPad Prism 6 (GraphPad Prism LLC.). Normality of the

data distribution was confirmed with Kolmogorov-Smirnov test. The results were expressed as mean ±

SD. All data were analyzed with Kruskal-Wallis test of one-way ANOVA, except for the decoy effect

test of E. coli O78: H11, which was done with RM one-way ANOVA. Significant difference was defined

as p<0.05 (*p<0.05), p<0.1 was considered as a statistical trend.

3. Results

3.1 HPAEC analysis of the hMOs and DFβCD

We first analyzed the chromatographic behavior of the compounds under study here. All compounds

were subjected to High-Performance Anion-Exchange Chromatography (HPAEC) coupled to a Pulsed

Amperometric Detector (PAD), and an overview of the respective masses and retention times (Rt) is

given in Table 1 and the chromatograms are shown in Figure S1. 2’-FL and 3-FL were analyzed using

gradient A, while βCD and difucosylated βCD (DFβCD) were analyzed using gradient B. Owing to the

different masses and interactions with the column, the retention times on the HPAEC column differed

greatly. The DFβCD sample was shown to contain 20% of monofucosylated βCD (MFβCD), which was

confirmed by comparing the retention time with that of a pure MFβCD sample. The HPAEC elution

pattern showed that DFβCD (16.7 min) was accompanied by a minor peak at 15.7 min (Figure S1),

presumably corresponding to an isomer with a different fucose substitution pattern.

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Table 1. Retention times of the hMOs and hMO mimics used in this study.

Compound Exact mass (Da) Rt (min)

2’-FL 488.1741 8.2

3-FL 488.1741 5.7 Gradient A Gradient

βCD 1134.3698 29.2

MFβCD 1280.4277 21.0

Gradient B Gradient DFβCD 1426.4856 15.7, 16.7

Gradient A: 0-15 % B (0-15 min), 15-100 % B (15-20 min), 100 % B (20-25 min), 0 % B (25-45 min);

Gradient B: 2.5-25 % B (0-30 min), 25-100 % B (30-40 min), 100 % B (40-45 min), 2.5 % B (45-60

min)

3.2 DFβCD is resistant to digestion by pancreatic enzymes

Human milk oligosaccharides are generally characterized as non-digestible carbohydrates because they

resist digestion by salivary α-amylase and digestive enzymes of the small intestine.[38, 39]

Consequently, a prerequisite for novel hMO-type compounds to exert beneficial effects in the intestinal

environment is that they also resist enzymatic digestion in the upper part of the gastrointestinal tract.[38]

Because DFβCD is a starch-like compound composed of -1,4-linked glucose units (Figure 1), it may

be digested by salivary α-amylase or pancreatic α-amylase in the small intestine. To determine the

resistance to digestion, DFβCD and β-cyclodextrin (βCD) were digested in an in vitro model according

to Martens et al.[40] and the amount of released glucose was quantified over time (Figure 2).

11 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399972; this version posted November 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Figure 2. In vitro digestibility of DFβCD (×) and βCD (●) during 240 min of incubation expressed as

free (released) glucose of total glucose (%). Soluble potato starch was digested for 90% within 120 min

of incubation (Figure S2). For reference, rapidly digestible starch (RDS), slowly digestible starch (SDS),

total digestible starch (TDS), and resistant starch (RS) are indicated in the figure.

The results show that DFβCD is almost fully resistant to digestion by pancreatic enzymes. Interestingly,

βCD was also quite resistant towards digestion, as only 10% digestion was observed within 120 min of

incubation, whereas the other 90% can be recognized as resistant starch according to the definition of

Englyst et al.[41] Apparently, the cyclic form has a large influence on the binding of the substrate by

digestive enzymes. For comparison, soluble potato starch was digested as a positive control, which was

90% hydrolyzed within 120 min of incubation (Figure S2). From the curve in Figure 2, it can be

expected that βCD would be fully degradable by pancreatic enzymes upon extended incubation times.

3.3 DFβCD is resistant to bacterial fermentation and does not induce short-chain fatty acid

production

Having established that DFβCD resists enzymatic digestion and is likely to arrive in the colon intact, we

subsequently tested the possible fermentability of DFβCD by infant microbiota. All compounds were

initially subjected to a fermentation experiment in vitro for 36 h using the fecal inoculum of a 9 month-

old infant, and the extent of fermentation over time was quantified by the disappearance of the compound

peak using HPAEC-PAD analysis (Figure 3A).

A B

Figure 3. Fermentation studies. A) HPAEC elution pattern of DFβCD and βCD at 0 and 24 h of in

vitro fermentation by 9-month old infant inoculum. Medium compounds are indicated with an *, malto-

oligomers are indicated with Glc, and MD2-7. B) Time-dependent in vitro fermentation of DFβCD (),

MFβCD (▲) and βCD () during 24 h.

Interestingly, whereas unsubstituted βCD was completely utilized after 24 h of fermentation, no

degredation was observed for DFβCD. A small decrease in the peak area of MFβCD was observed

between t = 0 h and t = 24 h, suggesting the fermentation was very slow (Figure 3A). As a comparison,

the fermentation experiment was also conducted on 2’-FL and 3-FL, and both hMOs were completely

degraded after 24 h of fermentation (Figure S3). Substrate degradation was monitored closely over time

to assess the fermentation kinetics (Figure 3B). DFβCD was indeed not degraded at all by microbial

enzymes of a 9-month old infant’s microbiome during batch fermentation. However, it seems that the

degradation of mono-fucosylated MFβCD, which is present for 20 % within the DFβCD mix, started

between 8-12 h of incubation reaching 70% fermentation after 24 h. Degradation of βCD started after

8 h of incubation quite suddenly and was completed at 12 h of incubation. No decrease in substrate

degradation was observed until 8 h of incubation and also no intermediate malto-oligomers were

13 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399972; this version posted November 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

detected by HPAEC-PAD at any time of incubation. This delay may suggest that opening of the CD ring

is the limiting step for successful degradation. 2’-FL and 3-FL were both degraded by the microbiota of

this 9-month old infant (Figure S3B), and the degradation of 2’-FL seemed to be slightly faster than

that of 3-FL.

After fermentation of the four compounds using a 9-month old infant inoculum, the production

of short-chain fatty acids (SCFA) was analyzed using HPLC-RI-UV (Figure S4). In the case of DFβCD,

little change in SCFA production compared to the fermentation control in medium was observed

(Figures S4A and S4E). This was as expected based on the observed lack of degradation of DFβCD

(Figure 3). Fermentation of unsubstituted βCD resulted in faster SCFA production compared to the

fermentation control, and a higher amount of butyrate was produced (acetate:butyrate 67:32 compared

to 80:20 at 12 h, Figures S4B and S4E). Fermentation of the human milk oligosaccharides 2’-FL and

3-FL also resulted in a quite fast production of acetate and intermediate acids lactate and succinate (for

2’-FL the ratio is acetate:butyrate:lactate:succinate 65:13:13:8, and for 3-FL the ratio is 67:11:14:7 at

12 h, Figures S4C and S4D), which were further converted to acetate and butyrate over the course of

the experiments.

3.4 DFβCD reveals structure-dependent anti-adhesive effects against E. coli O78:H11

To investigate the anti-adhesive properties of DFβCD in comparison with 2’-FL, 3-FL, first an adhesion

assay with the laboratory E. coli strain ET8 was performed. In this experiment, Caco-2 cells were pre-

incubated with the compounds at concentrations of 2, 5, and 10 mg/mL, followed by exposure to E. coli

ET8 bacteria and quantification of adhered bacteria (Figure S5). Interestingly, of all compounds tested

only DFβCD at 10 mg/mL significantly inhibited the adhesion of E. coli ET8 to intestinal epithelial

Caco-2 cells, with a reduction in adhesion of 60% compared to the non-treated control (Figure S5C,

p<0.05). Subsequently, ETEC O78:H11 (H10407) was selected as a clinically relevant pathogen to

assess the anti-adhesive capacity of DFβCD. We started by pre-incubating Caco-2 cells with 2’-FL, 3-

FL, βCD and DFβCD for 24 h, followed by the addition of ETEC bacteria (log phase). Bacterial adhesion

in the presence of DFβCD and control hMOs was determined by counting the CFU per mL of serially

14 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399972; this version posted November 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

diluted washes from Caco-2 monolayers, as compared to the control incubation (no additive). Using the

control reaction, the fraction of adhering bacteria was corrected for continuous bacterial growth in the

assay. As shown in Figure 4A, when the Caco-2 cells were pre-incubated with the molecules for 24 h

before E. coli O78:H11 infection, we observed that 2’-FL, 3-FL, βCD and DFβCD all reduced the

adhesion of E. coli O78:H11 to Caco-2 cells. The decrease in inhibition was calculated to be 37%, 43%,

34%, 16%, respectively, of which only the adhesion reduction of 3-FL achieved statistical significance

(p < 0.05). Both 2’-FL and βCD showed a trend of inhibition of adhesion (p values of 0.06 and 0.09,

respectively).

In contrast, when the ETEC bacteria were first pre-incubated with the compounds and

subsequently added to confluent Caco-2 cells, 2’-FL, 3-FL, and DFβCD all significantly inhibited the

adhesion of E. coli O78:H11 to Caco-2 cells, with a reduction of 30% (p < 0.05), 21% (p < 0.05), and

42% (p < 0.05), respectively (Figure 4B). Of all compounds tested here, DFβCD actually revealed the

highest inhibition of adhesion. These results are fucose-dependent, as the non-fucosylated βCD did not

significantly impact bacterial adhesion.

Figure 4. hMOs and DFβCD in 10 mg/mL inhibited adhesion of E. coli O78: H11 to intestinal

epithelial Caco-2 cells. Caco-2 cells were cultured in 24 well plates for 21 days. The tested molecules

of 2’-FL, 3-FL, βCD, and DFβCD either pre-incubated with Caco-2 cells for 24h (A) or pre-incubated

with E. coli O78: H11 for 2h (B). Cell culture medium without tested molecules was taken as control.

15 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399972; this version posted November 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

4. Discussion

The increasing evidence that human milk oligosaccharides inhibit bacterial infections has led to a surge

in interest to understand the exact structure-activity relationships of specific hMOs, and to develop

methods to generate these structures. Especially the fucosylated hMOs have been linked to anti-

pathogenic effects,[42, 43] and this may be a result of the central role of fucose on extracellular glycans

that are involved in a plethora of biological functions, including serving as anti-adhesion molecules for

pathogens.[44] As hMO structures in general, and multiply fucosylated hMOs specifically, are

challenging to produce on large scale and with high purity, there is a high demand for alternative

compounds that elicit similar effects.[45]

Here we report the biological evaluation of the novel hMO mimic di-fucosyl-β-cyclodextrin

(DFβCD). Using enzymatic digestion and in vitro fermentation analyses, we established that DFβCD is

resistant to digestive enzymes, and as a result is expected to reach the large intestine intact. Apparently,

the digestive enzymes, that generally hydrolyze -1,4-linked Glc units that are also present in DFβCD,

are blocked from action by the two Fuc units. As non-fucosylated βCD is only slowly digested, the

cyclic structure of βCD is also hypothesized to have a major impact on the resistance. This fits well with

earlier reports that βCD is only slowly digested by -amylase, while CD (six glucose units) is resistant

and CD (8 glucose units) is quickly digested.[46] Both 2’-FL, 3-FL, and βCD are efficiently fermented

by microbial enzymes, whereas DFβCD is resistant to bacterial fermentation. We hypothesize that the

resistance observed for DFβCD is a result of the specific positioning of the two Fuc units on opposite

sides of the βCD ring (specifically at the A and D position).[28] This may block the accessibility to the

microbial α-amylases sufficiently to resist fermentation, at least over the course of the 24 h incubation.

Overall SCFA production in all fermentated substrates after 24 h did not differentiate much from the

levels of SCFAs produced in the medium-only samples, presumably due to the low substrate

concentration used (2 mg/mL). [36] Slight differences in level and relative concentrations of the various

acids at 12 h of fermentation can be seen, and are substrate dependent. Fermentation experiments at

higher concentrations (e.g., 10 mg/mL) may provide a more accurate picture of the levels of SCFAs

16 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399972; this version posted November 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

produced with 2’-FL, 3-FL, and βCD over time. From the resistance to digestion observed with DFβCD

it is expected that for this compound the absence of additional SCFA production will be confirmed.

Excitingly, DFβCD revealed anti-adhesive properties against enterotoxigenic E. coli strain

O78:H11. There are two major mechanisms that may be at the basis of the anti-adhesive effect of

DFβCD, i.e. through modulating intestinal cell susceptibility to bacterial adhesion by changing receptor

expression levels, or through direct scavenging of bacteria by serving as decoy substrates. Both possible

mechanisms were investigated by pre-incubating the compounds with the Caco-2 cells or the bacterial

cells, respectively. Because the anti-adhesive effect of DFβCD was especially apparent when pre-

incubated with the bacteria prior to exposure to Caco-2 intestinal epithelial cells, we postulate that

DFβCD acts as a decoy substrate. Upon pre-incubation of E. coli O78:H11 with DFβCD, the hMO

mimic may saturate the receptors on the bacterial cell surface, and thereby prevent binding to the glycans

on Caco-2 intestinal cells. Such binding would require the pathogen to express specific receptors that

generally bind epithelial cell surface-associated glycans, but when confronted with DFβCD, may bind

this compound instead. When the Caco-2 intestinal cells were first pre-incubated with DFβCD, a less

pronounced effect was observed, suggesting that DFβCD has little impact on the expression of cell-

surface proteins involved in adhesion.

One of the first steps in pathogen colonization is adhesion to host cells, and its ability to adhere

is directly correlated with the pathogen’s capacity to invade and infect. Fucose-dependent pathogens

have been shown to be prevented by fucose-containing hMOs from adhering to mucosal membranes.[47]

Interfering with pathogen adhesion is therefore an effective strategy to prevent infection.[48] Whereas

natural hMOs are ideal candidates for anti-adhesive compounds, hMO mimics also have a large potential

to fill the current void between functional relevance and availability of the natural hMO compounds.

The biological activity of DFβCD serves as a strong proof-of-concept that hMO mimics are capable of

eliciting anti-adhesive effects similar to hMOs. The generation and evaluation of other fucosylated

structures will further our knowledge on the potential of fucosylated compounds to block bacterial

infections.

17 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399972; this version posted November 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Supporting Information

Supplementary figures are available in a separate file.

Author contributions

M.T.C.W., S.V., P.d.V. and H.A.S. conceived the project. S.V. generated the DFβCD compound. C.Ko.

performed the anti-adhesion studies. C.Kl. performed the digestion and fermentation studies. C.Ko.,

C.Kl, P.d.V., H.A.S., and M.T.C.W. were involved in data analysis. M.T.C.W. and S.V. wrote the

manuscript with input from all authors. All authors were involved in proofreading the manuscript.

Acknowledgments

This work was financially supported by the European Union through the Rosalind Franklin Fellowship

COFUND project 60021 (to M.T.C.W.), and by the China Scholarship Council (CSC) grant number

201600090212 (to C.Ko).

Conflict of interest

The authors declare no conflict of interest.

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